Carbohydrate-based synthetic vaccines for HIV

ABSTRACT

The present invention relates to a constructed oligosaccharide cluster, optionally bonded to an immunogenic protein, that can be administered to a subject to induce an immune response for increasing production of 2G12 and/or used in assays as reactive sites for determining compounds that inactivate and/or bind the high-mannose oligosaccharide cluster. Compositions comprising these clusters, methods of using these clusters and compositions are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. §371 andclaims the priority of International Patent Application No.PCT/US2003/032496 on Oct. 14, 2003, which in turn claims priority ofU.S. Provisional Patent Application No. 60/417,764 filed on Oct. 11,2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to vaccines, and moreparticularly, to an HIV vaccine comprising immunogenic high-mannose typeoligosaccharide clusters that mimics the HIV carbohydrate antigen havingan affinity for the HIV-1 neutralizing antibody 2G12.

2. Background of the Related Art

HIV is a member of the lentivirus family of retroviruses. Retrovirusesare small-enveloped viruses that contain a single-stranded RNA genome,and replicate via a DNA intermediate produced by a virally encodedreverse transcriptase, an RNA-dependent DNA polymerase.

The HIV viral particle comprises a viral core, composed in part ofcapsid proteins, together with the viral RNA genome and those enzymesrequired for early replicative events. A myristylated gag protein formsan outer shell around the viral core, which is, in turn, surrounded by alipid membrane envelope derived from the infected cell membrane. The HIVenvelope surface glycoproteins are synthesized as a single160-kilodalton precursor protein, which is cleaved by a cellularprotease during viral budding into two glycoproteins, gp41 and gp120.gp41 is a transmembrane glycoprotein and gp120 is an extracellularglycoprotein, which remains non-covalently associated with gp41,possibly in a trimeric or multimeric form.

Based on structural analysis, HIV-1 gp120 contains multiple high-mannosetype N-glycans. These discontinuous oligosaccharide chains are groupedtogether to form a unique oligosaccharide microdomain. This high-mannoseoligosaccharide grouping to form an epitope site has not been found inany human glycoproteins and is unique to HIV-1.

The worldwide epidemic of the human immuno-deficiency virus type 1(HIV-1) urges the development of an effective HIV vaccine. Yet, it hasbeen difficult to design effective immunogens that are able to elicitbroadly neutralizing antibodies against HIV-1 primary isolates. Inaddition to sequence variability of neutralizing epitopes, HIV-1 hasalso evolved other mechanisms to evade immune attack, including changeof conformations, shielding of conserved epitopes through heavyglycosylations, and formation of compact glycoprotein complexes(envelope spikes) that hinder the accessibility of epitopes to immuneresponses. It becomes clear that a successful strategy in developing aneffective HIV-1 vaccine relies on the identification of conservedepitopes on HIV-1 that are accessible to neutralization and on thedesign of epitope-based immunogens that stimulate high immune responses.

So far, only a few human monoclonal antibodies (MAbs) have beenidentified that are able to neutralize a broad range of HIV-1 primaryisolates. These include MAbs b12 and 2G12 that target the outer envelopeglycoprotein gp120, and MAbs 2F5 and 4E10 that target the inner envelopeglycoprotein gp41. The broadly neutralizing abilities of these MAbsimplicate the existence of conserved and accessible antigenicdeterminants, i.e., epitopes, on the surface of most HIV-1 primaryisolates. Passive immunization using these MAbs either alone or incombination has shown that these MAbs protect against HIV-1 challenge inanimal models when present at sufficient concentrations prior to orshortly after exposure(12). However, results have been limited anddeterminative by concentration and ongoing re-immunization.

Among the broadly HIV-1 neutralizing antibodies so far identified, thehuman monoclonal antibody 2G12 is the only one that directly targets thesurface carbohydrate antigen of HIV-1. Several pieces of evidencesuggest that the epitope of 2G12 is a unique cluster of high-mannosetype oligosaccharides (oilgomannose) on HIV-1 gp120. Initial mutationalstudies indicated that the oligomannose sugar chains at theN-glycosylation sites N295, N332, N339, N386, N392, and N448 might beinvolved in 2G12 recognition (9). Two recent studies further proposedthat the epitope of 2G12 might consist of several Manα1-2Man-linkedmoieties contributed by the oligomannose sugar chains at sites N295,N332, and N392 that form a unique cluster on gp120 (81, 82).

However, HIV-1 gp120 expresses an array of high-mannose oligosaccharidesranging from Man₅, Man₆, to Man₉ on these sites (76-78). These diverseoligomannose glycoforms of the 2G12 epitope on HIV-1 gp120 are likely todilute any potential immune response to the epitope. This may partiallyexplain why gp120 itself raises a limited number of 2G12-likeantibodies. Further, carbohydrates themselves are generally poorimmunogens, which may explain why 2G12-like neutralizing antibodies arerare in natural infection. Thus, it would be advantageous to provide arepresentative carbohydrate structure that would increase production of2G12 neutralizing antibodies and that could be used as a component in atherapeutic composition.

SUMMARY OF THE INVENTION

The present invention relates to a constructed oligosaccharide cluster,optionally bonded to an immunogenic protein, that can be administered toa subject to induce an immune response for increasing production ofneutralizing antibodies, such as 2G12, that bind to a conserved clusterof oligosaccharide sugars on gp120 and/or used in assays as reactivesites for determining compounds that inactivate and/or bind the aconserved cluster of oligosaccharide sugars on gp120.

In one aspect, the present invention relates to at least onehigh-mannose oligosaccharide positioned on a scaffolding framework ormolecule that is conjugated to an immunogenic protein to form ahigh-mannose oligosaccharide/protein cluster thereby generating animmune enhancing vaccine.

In another aspect, the present invention relates to a novel high-mannoseoligosaccharide cluster comprising at least one high-mannoseoligosaccharide assembled on a monosaccharide scaffold to provide thefirst generation of novel, carbohydrate-based HIV-1 vaccine.

In yet another aspect, the present invention relates to a vaccinecomprising an oligosaccharide cluster covalently attached to ascaffolding framework, which in turn is conjugated to an immunogenicprotein. The general design of such a vaccine is shown in FIG. 2, whereMan₉ represents the major high-mannose type oligosaccharide structurefound on HIV-1 gp120, and the immunogenic protein can be any potentimmune-stimulating carrier protein such as KLH (keyhole limpethemocyanin). The number of the oligosaccharide chains attached to thescaffold could be 2, 3, 4, or more.

Another aspect of the present invention relates to methods forgenerating an oligosaccharide cluster comprising the steps of:

-   -   covalently linking or attaching at least one high-mannose        oligosaccharide chain to a scaffold molecule to generate an        oligosaccharide cluster that mimics an antigenic structure        having affinity for 2G12 antibodies. The high-mannose        oligosaccharide chains may be obtained from the digestion of        soybean agglutinin or produced by chemical synthesis.        High-mannose oligosaccharide chains can include any structural        variant of Man₉ (containing 9 mannose residues), Man₈:, Man₇,        Man₆, Man₅ or a combination thereof Any combination of these        high-mannose oligosaccharide chains may be attached to a        scaffolding framework which may include, but is not limited to,        monosaccharides, cyclic peptides, cyclic organic compounds, or        compounds such as 11-bis-maleimidetetraethyleneglycol.

In yet another aspect, the present invention relates to antibodies,including polyclonal and monoclonal, and production thereof, wherein theantibody is immunoreactive with an oligosaccharide cluster and/or anoligosaccharide/protein cluster of the present invention.

In still a further aspect, the present invention contemplates a processfor producing an antibody, which is immunoreactive with anoligosaccharide cluster and/or an oligosaccharide/protein cluster of thepresent invention comprising the steps of:

-   -   (a) introducing the oligosaccharide cluster and/or the        oligosaccharide/protein cluster into a live animal subject; and    -   (b) recovering antisera comprising antibodies specific for the        oligosaccharide cluster and/or the oligosaccharide/protein        cluster.

Another aspect relates to a diagnostic testing system for detectingHIV-1 infection, the testing system comprising:

-   -   contacting a biological sample being tested for occurrence of        HIV-1 virus with antisera specific for a high-mannose        oligosaccharide cluster of the present invention that mimics a        carbohydrate antigenic structure having affinity for 2G12        antibodies; and determining binding between the antisera and the        biological sample.

In another aspect, the present invention contemplates a diagnostic kitfor detecting the presence of 2G12 antibodies in a biological sample,wherein the kit comprises a first container containing anoligosaccharide cluster of the present invention capable ofimmunoreacting with a 2G12 neutralizing antibody in the biologicaltesting sample. Preferably, the kit of the invention further comprises asecond container containing a second antibody with an indicator thatimmunoreacts with a binding antibody to the oligosaccharide cluster ofthe present invention.

Alternatively, the present invention provides a process for detectingcandidate compounds that potentially interact with a conserved clusterof oligomannose sugars on gp120, the process comprising:

-   -   contacting the candidate compound with an oligosaccharide        cluster and/or an oligosaccharide/protein cluster of the present        invention; and    -   determining the binding affinity of the candidate compound for        the oligosaccharide cluster and/or an oligosaccharide/protein        cluster of the present invention.

Another aspect of the present invention relates to a method to induceproduction of neutralizing 2G12 antibodies, the method comprising:

-   -   administering to a subject a composition comprising an        oligosaccharide cluster and/or an oligosaccharide/protein        cluster of the present invention in an effective amount to        induce production of neutralizing 2G12 antibodies.

In still another aspect, the present invention relates to a method oftreating an HIV-1 virus infection, comprising:

-   -   administering to a patient a composition comprising a        therapeutically effective amount of the oligosaccharide cluster        and/or an oligosaccharide/protein cluster to induce prolonged        production of neutralizing antibodies, wherein the neutralizing        antibodies have an affinity for a conserved cluster of        oligosaccharide sugars on gp120.

Yet another aspect relates to a method of making a high-mannoseoligosaccharide/protein cluster comprising the steps of: a) covalentlyattaching high-mannose oligosaccharides to a scaffolding molecule toform the oligosaccharide cluster; and b) covalently attaching animmunogenic carrier protein to the oligosaccharide cluster to form thehigh-mannose oligosaccharide/protein cluster.

The high-mannose oligosaccharide/protein cluster of the presentinvention may be administered alone or in a pharmaceutical compositionas a vaccine in a therapeutically effective amount to elicit an enhancedimmune response or a protective immune response in an animal.

The compositions of the present invention may further comprise at leastone antiviral agent. The antiviral agent may include any agent thatinhibits entry into a cell or replication therein of an infectiousvirus, and specifically retroviruses, such as HIV viruses. The antiviralagents include, but not limited to nucleoside RT inhibitors, CCR5inhibitors/antagonists, viral entry inhibitors and their functionalanalogs.

The pharmaceutical compositions may be administered alone or incombination with a therapeutically effective amount of at least oneantiviral agent, including, but not limited to:

nucleoside RT inhibitors, such as Zidovudine (ZDV, AZT), Lanivudine(3TC), Stavudine (d4T), Didanosine (ddl), Zalcitabine (ddC), Abacavir(ABC), Emirivine (FTC), Tenofovir (TDF), Delaviradine (DLV), Efavirenz(EFV), Nevirapine (NVP), Fuzeon (T-20), Saquinavir (SQV), Ritonavir(RTV), Indinavir (IDV), Nelfinavir (NFV), Amprenavir (APV), Lopinavir(LPV), Atazanavir, Combivir (ZDV/3TC), Kaletra (RTV/LPV), Trizivir(ZDV/3TC/ABC);

CCR5 inhibitors/antagonists, such as SCH-C, SCH-D, PRO 140, TAK 779,TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies;

viral entry inhibitors, such as Fuzeon (T-20), NB-2, NB-64, T-649,T-1249, SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602,UK-427, 857; and functional analogs or equivalents thereof.

Yet still another aspect relates to a method of increasing the affinityof epitope mimics of the present invention to gp120 comprisingmanipulating the spatial orientation of high-mannose oligosaccharidechains on a scaffolding framework to create antibodies exhibitinghigh-affinity multivalent interaction with a conserved cluster ofoligomannose sugars on gp120.

These and other aspects of the present invention, will be apparent fromthe detailed description of the invention provided hereinafter

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates Mab 2G12 as described in the prior art by Trkola, etal 1996.

FIG. 2 illustrates the general structure of the conjugate vaccine of thepresent invention.

FIG. 3 shows the acetylation of Man₉GlCNAc₂ASn.

FIG. 4 shows a synthesis scheme of a galactose-based template forattachment of high-mannose oligosaccharide chains.

FIG. 5 shows assembly of high-mannose oligosaccharide chains onto agalactose-based template.

FIG. 6 shows conjugation of the oligosaccharide cluster to a carrierprotein.

FIG. 7 shows conjugation of a high-mannose oligosaccharide chain to acarrier protein.

FIG. 8 shows the structure of a high-mannose oligosaccharide cluster.

FIG. 9 shows the introduction of a sulfhydryl group onto Man₉GlcNAc₂Asn.Reaction conditions: (a) phosphate buffer (pH 7.4) containing 30% MeCN,r.t., 2 h; (b) hydroxylamine (0.5 M), EDTA (25 mM) in phosphate buffer(pH 7.5), r.t., 1 h.

FIG. 10 shows ligation between the maleimide cluster 5 and the thiololigosaccharide derivative 4. Reaction conditions: (a) phosphate buffer(pH 6.6, 50 mM) containing 40% MeCN, r.t., 1 h.

FIG. 11 shows ligation between the bivalent scaffold 7 and the thiololigosaccharide derivative 4. Reaction conditions: (a) phosphate buffer(pH 6.6), r.t., 1 h.

FIG. 12 shows ESI-MS and HPLC profile of the tetra-Man9 cluster (i.e.,Tetra-Man9, as shown in FIG. 15).

FIG. 13 shows structures of typical HIV-1 high-mannose oligosaccharides.

FIG. 14 shows inhibition of 2G12 binding to gp120 by high-mannose typeoligosaccharides of the present invention. 2G12 binding (%) was plottedagainst the log of competing carbohydrate concentrations in micromolarunits. Triangle, Man₅GlcNAc; solid circle, Man₆GlcNAc; solid square,Man₉GlcNAc.

FIG. 15 shows structures of galactose-based maleimide clusters andsynthetic oligomannose clusters.

FIG. 16 shows inhibition of 2G12 binding to gp120 by syntheticoligomannose clusters. 2G12 binding (%) was plotted against the log ofcompeting carbohydrate concentrations in micromolar units. Soliddiamond, Man₉GlcNAc₂Asn; open triangle, Man₉-dimer; solid triangle,Bi-Man₉; open circle,

DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate review of the various embodiments of theinvention and provide an understanding of the various elements andconstituents used in making and using the present invention, thefollowing terms used in the invention description have the followingmeanings.

Definitions

The term “oligosaccharide cluster,” as used herein, is a scaffoldcomprising at least one high-mannose oligosaccharide chain.

The term “oligosaccharide/protein cluster,” as used herein means ahigh-mannose oligosaccharide cluster attached to an immune stimulatingcarrier protein.

The term “immunogenic protein,” as used herein, means a protein suitablefor conjugation to the oligosaccharide cluster including, but notlimited to keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid,bovine serum albumin, ovalbumin, thyroglobulin, myoglobin, cholera toxinβ-subunit, immunoglobulin and/or tuberculosis purified proteinderivative.

The term “scaffold or scaffolding,” as used herein means a structurewhereby oligosaccharide chains are attached, wherein the structure mayinclude, but is not limited to monosaccharides, cyclic peptides and/orcyclic organic compounds.

A method of treating a viral infection is meant herein to include“prophylactic” treatment or “therapeutic” treatment. A “prophylactic”treatment is a treatment administered to a subject who does not exhibitsigns of a disease or who exhibits early signs of the disease for thepurpose of decreasing the risk of developing pathology associated withthe disease.

The term “therapeutic,” as used herein, means a treatment administeredto a subject who exhibits signs of pathology for the purpose ofdiminishing or eliminating those signs.

The term “therapeutically effective amount,” as used herein means anamount of compound that is sufficient to provide a beneficial effect tothe subject to which the compound is administered. A beneficial effectmeans rendering a virus incompetent for replication, inhibition of viralreplication, inhibition of infection of a further host cell, orincreasing CD4 T-cell count, for example.

The term “specific binding,” as used herein, in reference to theinteraction of an antibody and a protein or peptide, means that theinteraction is dependent upon the presence of a particular structure(i.e., the antigenic determinant or epitope) on the protein; in otherwords, the antibody is recognizing and binding to a specific proteinstructure rather than to proteins in general.

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant.

THE INVENTION

Oligosaccharide Clusters and Oligosaccharide/Immunogenic ProteinClusters

The present invention relates to high-mannose oligosaccharide clusterscomprising at least one high-mannose oligosaccharide covalently attachedor linked to a scaffold thereby forming a high-mannose oligosaccharidecluster. The high-mannose oligosaccharide is selected from the groupconsisting of Man₉ Man₈, Man₇, Man₆, Man₅ and any combination thereofThe high-mannose oligosaccharide can be isolated from soybean agglutininor synthesized by techniques well known to one skilled in the art.Preferably, the scaffold framework comprises at least two high-mannoseoligosaccharides, and more preferably, four Man₉ are covalently linkedto the scaffolding.

The present invention further comprises linking the high-mannoseoligosaccharide cluster to an immunogenic protein thereby inducingproduction of antibodies having an affinity for the high-mannoseoligosaccharide cluster. Preferably, the high-mannose oligosaccharidecluster comprises at least two mannose oligosaccharides covalentlyattached to a monosaccharide scaffold and the oligosaccharide cluster iscovalently attached to keyhole limpet hemocyanin acting as theimmunogenic protein.

The high-mannose oligosaccharide clusters andoligosaccharide/immunogenic protein clusters of the present inventionmay be administered as vaccines with various pharmaceutically acceptablecarriers. Pharmaceutically acceptable carriers includes those approvedfor use in animals and humans and include diluents, adjuvants,excipients or any vehicle with which a compound, such as multivalentpeptides and/or maleimide clusters, is administered. Pharmaceuticallyacceptable carriers include but are not limited to water, oils, saline,dextrose solutions, glycerol solutions, excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,powdered non-fat milk, propylene glycol and ethanol. Pharmaceuticalcompositions may also include wetting or emulsifying agents, or pHbuffering compounds.

It becomes clear that a successful strategy in developing an effectiveHIV-1 vaccine relies on the identification of conserved epitopes onHIV-1 that are accessible to neutralization and on the design ofepitope-based immunogens that stimulate high immune responses. Insearching for conserved and accessible antigenic structures for vaccinedesign, a well known and yet not adequately exploited target, thesurface carbohydrate structures of HIV-1 gp120 was selected.

Based on structural analysis, HIV-1 gp120 contains multiple high-mannosetype N-glycans. These discontinuous oligosaccharide chains are clusteredtogether to form a unique oligosaccharide microdomain. The high-mannoseoligosaccharide cluster has not been found in any glycoproteins and isunique to HIV-1. In addition, recent studies suggest that thehigh-mannose oligosaccharide cluster may constitute the actual epitopesof the broadly neutralizing 2G12.

Pharmaceutical Compositions

The present invention provides for compositions comprising at least onehigh-mannose oligosaccharide complex or high-mannoseoligosaccharide/protein complex and optionally at least one antiviralagent, as well as methods of enhancing an immune response therebyinducing increased production of neutralizing HIV antibodies fortreating and/or reducing the effects of HIV. The methods compriseadministering said compositions comprising the one high-mannoseoligosaccharide complex or high-mannose oligosaccharide/protein complexand optionally antiviral agents, wherein the two compounds can beadministered, separately, simultaneously, concurrently or consecutively.

Anti-Viral Compounds

In one aspect the compositions and methods of the present invention mayfurther comprise a therapeutically effective amount of at least oneantiviral agent, including, but not limited to nucleoside RT inhibitors,CCR5 inhibitors/antagonists, viral entry inhibitors and functionalanalogs thereof.

Preferably, the antiviral agent comprises nucleoside RT inhibitors, suchas Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T), Didanosine(ddl), Zalcitabine (ddC), Abacavir (ABC), Emirivine (FTC), Tenofovir(TDF), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Fuzeon(T-20), Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfinavir(NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir(ZDV/3TC), Kaletra (RTV/LPV), Trizivir (ZDV/3TC/ABC);

CCR5 inhibitors/antagonists, such as SCH-C, SCH-D, PRO 140, TAK 779,TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies;

viral entry inhibitors, such as Fuzeon (T-20), NB-2, NB-64, T-649,T-1249, SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602,UK-427, 857; and functional analogs thereof

Methods for Preventing and/or Treating a Viral Infection

The compositions and methods of the present invention can be used totreat or reduce effects of HIV viral infection in a subject potentiallyexposed to the infection. At least one high-mannose oligosaccharidecomplex or high-mannose oligosaccharide/protein complex of the presentinvention may be administered for the treatment of HIV either as singletherapeutic agents or when used in combination with antiretroviraldrugs.

A composition of the present invention is typically administeredparenterally in dosage unit formulations containing standard, well-knownnontoxic physiologically acceptable carriers, adjuvants, and vehicles asdesired. The term parenteral as used herein includes intravenous,intramuscular, intraarterial injection, or infusion techniques.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.Preferred carriers include neutral saline solutions buffered withphosphate, lactate, Tris, and the like.

The compositions of the invention are administered in substantiallynon-toxic dosage concentrations sufficient to ensure the release of asufficient dosage unit of the present complexes into the patient toprovide the desired inhibition of the HIV virus. The actual dosageadministered will be determined by physical and physiological factorssuch as age, body weight, severity of condition, and/or clinical historyof the patient. The active ingredients are ideally administered toachieve in vivo plasma concentrations of an antiviral agent of about0.01 uM to about 100 uM, more preferably about 0.1 to 10 uM, and mostpreferably about 1-5 uM, and of a high-mannose oligosaccharide complexor high-mannose oligosaccharide/protein complex of about 1 u.M-25 uM,more preferably about 2-20 uM, and most preferably about 5-10 uM. Itwill be understood, however, that dosage levels that deviate from theranges provided may also be suitable in the treatment of a given viralinfection.

Therapeutic efficacy of the high-mannose oligosaccharide complexes orhigh-mannose oligosaccharide/protein complexes can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining The LD₅₀ (The Dose Lethal To 50% Of ThePopulation) and The ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds, which exhibit large therapeutic indexes, are preferred. Thedata obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Further, the therapeutic compositions according to the present inventionmay be employed in combination with other-therapeutic agents for thetreatment of viral infections or conditions. Examples of such additionaltherapeutic agents include agents that are effective for the treatmentof viral infections or associated conditions such as immunomodulatoryagents such as thymosin, ribonucleotide reductase inhibitors such as2-acetylpyridine 5-[(2-chloroanilino) thiocarbonyl)thiocarbonohydrazone, interferons such as alpha-interferon,1-beta-D-arabinofuranosyl-5-(1-propynyl)uracil,3′-azido-3′-deoxythymidine, ribavirin and phosphonoformic acid.

In still another embodiment, the present invention provides antibodiesimmunoreactive with the high-mannose oligosaccharide complexes orhigh-mannose oligosaccharide/protein complexes of the present invention.The antibodies may include both monoclonal and polyclonal.

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a high-mannose oligosaccharide complex orhigh-mannose oligosaccharide/protein complex of the present invention,and collecting antisera from that immunized animal. A wide range ofanimal species can be used for the production of antisera. Typically ananimal used for production of antisera is a rabbit, a mouse, a rat, ahamster or a guinea pig. Because of the relatively large blood volume ofrabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

Exemplary and preferred immunogenic proteins are keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such asovalbumin, mouse serum albumin or rabbit serum albumin can also be usedas carriers. Means for conjugating the high-mannose oligosaccharidecomplex or high-mannose oligosaccharide/protein complex are well knownin the art and include glutaraldehyde, Mmaleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

Immunogenicity to a particular immunogen can be enhanced by the use ofnon-specific stimulators of the immune response known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant,incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

The amount of immunogen used for the production of polyclonal antibodiesvaries inter alia, upon the nature of the immunogen as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies is monitored by sampling blood of the immunized animal atvarious points following immunization. When a desired level ofimmunogenicity is obtained, the immunized animal can be bled and theserum isolated and stored.

Typically, a monoclonal antibody of the present invention can be readilyprepared by a technique which involves first immunizing a suitableanimal with a selected antigen (e.g., the high-mannose oligosaccharidecomplexes or high-mannose oligosaccharide/protein complexes of thepresent invention) in a manner sufficient to provide an immune response.Rodents such as mice and rats are preferred animals. Spleen cells fromthe immunized animal are then fused with cells of an immortal myelomacell. Where the immunized animal is a mouse, a preferred myeloma cell isa murine NS-1 myeloma cell.

The fused spleen/myeloma cells are cultured in a selective medium toselect fused spleen/myeloma cells from the parental cells. Fused cellsare separated from the mixture of non-fused parental cells, for example,by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media. Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, the mediais supplemented with hypoxanthine. This culturing provides a populationof hybridomas from which specific hybridomas are selected. Typically,selection of hybridomas is performed by culturing the cells bysingle-clone dilution in microliter plates, followed by testing theindividual clonal supernatants for reactivity with the antigenicoligosaccharide complexes. The selected clones can then be propagatedindefinitely to provide the monoclonal antibody.

By way of specific example, to produce an antibody of the presentinvention, mice are injected intraperitoneally with between about 1-200ug of an antigen comprising the high-mannose oligosaccharide complex orhigh-mannose oligosaccharide/protein complex of the present invention.At some time (e.g., at least two weeks) after the first injection, miceare boosted by injection with a second dose of the antigen andoptionally mixed with incomplete Freund's adjuvant. A few weeks afterthe second injection, mice are tail bled and the sera titered byimmunoprecipitation against radiolabeled antigen. Preferably, theprocess of boosting and titering is repeated until a suitable titer isachieved. The spleen of the mouse with the highest titer is removed andthe spleen lymphocytes are obtained by homogenizing the spleen with asyringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

Myeloma cells are combined under conditions appropriate to foster fusionwith the normal antibody-producing cells from the spleen of the mouse orrat injected with the antigen/oligosaccharide complexes of the presentinvention. Fusion conditions include, for example, the presence ofpolyethylene glycol. The resulting fused cells are hybridoma cells. Likemyeloma cells, hybridoma cells grow indefinitely in culture. Hybridomacells are separated from unfused myeloma cells by culturing in aselection medium such as HAT media (hypoxanthine, aminopterin,thymidine).

Unfused myeloma cells lack the enzymes necessary to synthesizenucleotides from the salvage pathway because they are killed in thepresence of aminopterin, methotrexate, or azaserine. Unfused lymphocytesalso do not continue to grow in tissue culture. Thus, only cells thathave successfully fused (hybridoma cells) can grow in the selectionmedia. Each of the surviving hybridoma cells produces a single antibody.These cells are then screened for the production of the specificantibody immunoreactive with an antigen/oligosaccharide complex of thepresent invention. Single cell hybridomas are isolated by limitingdilutions of the hybridomas. The hybridomas are serially diluted manytimes and, after the dilutions are allowed to grow, the supernatant istested for the presence of the monoclonal antibody. The clones producingthat antibody are then cultured in large amounts to produce an antibodyof the present invention in convenient quantity.

Screening Assays

In yet another aspect, the present invention contemplates a process ofscreening substances for their ability to interact with a conservedepitopic cluster of oligosaccharide sugars on gp120 and created mimicsof such an epitope including the high-mannose oligosaccharide complexesof the present invention, the process comprising the steps of providinga high-mannose oligosaccharide complex of the present invention andtesting the ability of selected test substances to interact with thathigh-mannose oligosaccharide complexes of the present invention.

The methods of the present invention make it possible to produce largequantities of a high-mannose oligosaccharide complex that mimics anepitope that immunoreacts with Mab 2G12 or antibodies reactive therewithfor use in screening assays.

Screening assays of the present invention generally involve determiningthe ability of a candidate test substance to bind to the high-mannoseoligosaccharide complexes of the present invention. These high-mannoseoligosaccharide complexes can be coupled to a solid support. The solidsupport can be agarose beads, polyacrylamide beads, polyacrylic beads orother solid matrices capable of being coupled to proteins. Well knowncoupling agents include cyanogen bromide, carbonyidiimidazole, tosylchloride, and glutaraldehyde.

Alternatively, the present invention provides a process of detecting HIVinfection, wherein the process comprises immunoreacting the biologicalsamples comprising suspected HIV virus with antibodies generated andhaving affinity for the high-mannose oligosaccharide complexes of thepresent invention (mimicking a conserved cluster of oligosaccharidesugars on gp120) to form an antibody-polypeptide conjugate and detectingthe conjugates.

A biological sample to be screened can be a biological fluid such asextracellular or intracellular fluid or a cell or tissue extract orhomogenate. A biological sample can also be an isolated cell (e.g., inculture) or a collection of cells such as in a tissue sample orhistology sample. A tissue sample can be suspended in a liquid medium orfixed onto a solid support such as a microscope slide.

In accordance with a screening assay process, a biological sample isexposed to an antibody immunoreactive with the 2G12 epitope located ongp120 of HIV. Typically, the biological sample is exposed to theantibody under biological reaction conditions and for a period of timesufficient for antibody-epitope conjugate formation. Biological reactionconditions include ionic composition and concentration, temperature, pHand the like. Ionic composition and concentration can range from that ofdistilled water to a 2 molal solution of NaCl. Temperature preferably isfrom about 25° C. to about 40° C. pH is preferably from about a value of4.0 to a value of about 9.0, more preferably from about a value of 6.5to a value of about 8.5 and, even more preferably from about a value of7.0 to a value of about 7.5. The only limit on biological reactionconditions is that the conditions selected allow forantibody-polypeptide conjugate formation and that the conditions do notadversely affect either the antibody or the peptide.

Exposure time will vary inter alia with the biological conditions used,the concentration of antibody and peptide and the nature of the sample(e.g., fluid or tissue sample). Means for determining exposure time arewell known to one of ordinary skill in the art. Typically, where thesample is fluid and the concentration of peptide in that sample is about10⁻¹⁰ M, exposure time is from about 10 minutes to about 200 minutes.

The presence of a gp120 in the biological sample is detected bydetecting the formation and presence of antibody-peptide conjugates.Means for detecting such antibody-antigen (e.g., peptide) conjugates orcomplexes are well known in the art and include such procedures ascentrifugation, affinity chromatography and the like, binding of asecondary antibody to the antibody-candidate peptide complex.

In one embodiment, detection is accomplished by detecting an indicatoraffixed to the antibody. Exemplary and well known such indicatorsinclude radioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), a second antibody oran enzyme such as horse radish peroxidase. Means for affixing indicatorsto antibodies are well known in the art and available in commercialkits.

EXPERIMENTAL PROCEDURES EXAMPLE 1

The HIV-1 envelope glycoprotein gp120 is important target for HIV-1vaccine design, although it has been difficult to design effectiveimmunogens that elicit neutralizing antibodies reactive to a broad rangeof HIV-1 primary isolates (1-3).

In searching for conserved and accessible antigenic structures forvaccine design, attention was turned to the not adequately exploitedtarget, the surface carbohydrate structures of HIV-1 gp120. HIV-1 gp120contains high numbers of high-mannose type N-glycans, most of which areconserved among HIV-1 isolates. Molecular modeling studies suggest thatthese otherwise discontinuous carbohydrate moieties are clusteredtogether on folded gp120 to form a unique oligosaccharide microdomain.In addition, the discontinuous epitopes of the broadly neutralizingantibody 2G12 were mapped in the high-mannose N-glycosylation sites.

The goal of the present invention was to develop an effective anti-HIVvaccine through targeting unique carbohydrate structures present onHIV-1 and incorporating this novel carbohydrate antigenic structure intoan HIV-1 vaccine. The general structure of such a vaccine is shown inFIG. 2, where Man₉ represents the major high-mannose typeoligosaccharide structure found on HIV-1 gp120, and KLH (keyhole limpethemocyanin) is a potent immune-stimulating carrier protein.

A novel high-mannose oligosaccharide cluster is assembled using ascaffold approach that has been previously used for constructingmultivalent peptides (43). As stated above, carbohydrates themselves aregenerally poor immunogens, which may explain why 2G12-like neutralizingantibodies are rare in natural infection. However, conjugation of thedesigned carbohydrate antigen to an immunogenic protein such as KLH hasthe ability to render the designed carbohydrate antigen highlyimmunogenic.

Carbohydrate-based conjugate vaccines have been developed for elicitingprotective immune responses against pathogens such as bacteria (53).However, heretofore carbohydrate antigens have not been adequatelyexploited for HIV-1 vaccine design, despite their abundance on the HIV-1surface.

The synthesis of the designed carbohydrate-based conjugate vaccinerequires a relatively large quantity of the Man₉ oligosaccharides.Man₉GlcNAc2Asn was originally prepared from soybean agglutinin (SBA)through pronase digestion for structural analysis (54, 55).Man₉GlcNAc₂Asn from SBA has been used for chemoenzymatic synthesis ofhigh-mannose type glycopeptides (56, 57). For the purpose ofsynthesizing the carbohydrate-conjugate vaccine, a modified procedurehas been established that allows for the efficient preparation ofMan₉GlcNAc₂Asn on a relatively large scale.

Crude SBA was obtained by fractional precipitation of non-processedsoybean flour (Sigma) with ammonium sulfate (55-65%). The crude SBA wasthen subject to thorough digestion with pronase (Sigma).High-Performance Anion-Exchange chromatography (HPAEC) with PulsedElectrochemical Detection (PED) was used to monitor the digestionprocess. A 48 h digestion led to the conversion of only half of the Man₉oligosaccharide into the form of Man₉GlcNAc₂Asn; the rest are in theforms of glycopeptides with several amino acid residues attached, whichare relatively difficult to digest. Complete digestion ofprotein/peptides was achieved through adding extra portions of pronaseat intervals and using elongated digestion time (7 days). The finalproduct, Man₉GlcNAc₂Asn, was readily isolated by gel filtrationchromatography on a column of SEPHADEX G-50 with 0.1 M acetic acid asthe eluent. The isolated product was characterized by ¹H-NMR, ESI-MS,and carbohydrate compositional analysis. From 2 kg of soybean flour,about 180 mg of pure Man₉GlcNAc₂Asn was obtained and the preparation canbe readily scaled up. Next, we selectively protected the free aminogroup of Asn by reacting Man₉GlcNAc₂Asn with acetic anhydride in aqueoussodium bicarbonate to give the acetylated Man₉GlcNAc₂Asn (1) in 85%yield (FIG. 3). Compound 1, with a free carboxyl group in the molecule,is now suitable for coupling to a scaffold to form a clusteredhigh-mannose oligosaccharide structure.

Reliable methods have been established for selective modification ofmonosaccharides, which have been used as scaffolds (templates) toassemble multivalent gp41 peptides (43). Compared with other scaffoldmolecules, monosaccharides have a rigid ring structure and allow thedisplay of the antigenic structures in a defined, three-dimensionalformat. The sugar-scaffold approach is used for the synthesis of thedisclosed high-mannose oligosaccharide cluster.

A galactose-based template is synthesized, compound 6, which containsfour amino functionalities on the arms that are arranged in a clusteredformat and are suitable for attachment of four copies of high-mannoseoligosaccharide chains. In addition, compound 6 has a carboxylfunctionality in the aglycon portion that is used for selectiveconjugation to a carrier protein (FIG. 4).

To prepare compound 6 as shown in FIG. 4, a precursor compound (4) issynthesized. Briefly, an azido functionality was introduced in theaglycon portion of galactose in two steps: 1) refluxing galactose inchloroethanol to form chloroethyl α-galactoside and 2) substitution ofthe chloro atom with sodium azide to give compound 2 in 78% yield fromgalactose. Allylation of compound 2 with allyl bromide/NaH in DMFafforded compound 3 in 88% yield. Reduction of the azido group incompound 3 with triphenylphosphine gave compound 4 in 45% yield. All thecompounds were purified by silica gel chromatography and characterizedby NMR and MS. To complete the synthesis of compound 6, compound 4 iscoupled with succinic acid monomethyl ester to provide compound 5. Fouramino functionalities are then introduced by photoaddition of cysteamineto the allyl groups to give the template 6 (FIG. 4). Photoaddition ofthiols to allyl groups is a very mild reaction for functional grouptransformations and we have previously used this reaction to preparecyclodextrin-based polyamines and monosaccharide templates formultivalent peptide assembling (43, 58).

Various methods for the coupling of compound 6 with the N-acetylatedMan₉GlcNAc₂Asn (1), which is prepared as described for (FIG. 3), areavailable (FIG. 5). HBTU is used as a coupling reagent. HBTU is apowerful coupling reagent for peptide bond formation and wassuccessfully used for coupling large, unprotected oligosaccharideglycosylamine with carboxyl groups in peptides (59, 60). The couplingreagent such as 1-ethyl-3-(3dimethylaminopropyl) carbodiimidehydrocholoride (EDC) is used to establish optimized conditions forassembly of the cluster. The reactions are monitored using HPLC analysis(reverse phase and size-exclusion).

Conjugation of synthetic carbohydrate antigens to an immune-stimulatingcarrier protein was accomplished by reductive amination which was shownto be a reliable method for conjugation. Reductive amination is used toconjugate the high-mannose oligosaccharide cluster to KLH. An aldehydefunctionality is introduced into the high-mannose oligosaccharidecluster 7. This is achieved through several steps of chemicaltransformations of 7 (FIG. 6).

First, the ester functionality in compound 7 is hydrolyzed to provide afree carboxylic acid, which is then reacted with 2-aminoacetaldhydedibenzyl acetal to give compound 8. The benzyl groups in compound 8 areselectively removed by palladium catalyzed hydrogenation to givecompound 9, with a free aldehyde functionality in the molecule. Finallyreductive amination between compound 9 and KLH is performed with sodiumcyanoborohydride (NaCNB113) in a phosphate buffer. The conjugate-vaccine10a is isolated by dialysis followed by lyophilization. The ratio ofantigen to carrier protein is determined by carbohydrate analysis andprotein assay. In addition, the coupling of aldehyde 9 to human serumalbumin (HSA) is performed in the same way to provide thecarbohydrate-HSA conjugate (FIG. 6). Free carbohydrate antigens aredifficult to immobilize in ELISA wells because of their very lowaffinity to plastic surface. The carbohydrate-HSA conjugate 10b is usedas a coating antigen for evaluating immune responses in ELISAs.

For comparative studies, a high-mannose oligosaccharide antigen, theN-acetylated Man₉GlcNAc₂Asn (1), is directly conjugated to KLH or HSA(FIG. 7). The aldehyde functional group is introduced into compound 1 intwo steps: reaction of compound 1 with 2-aminoacetaldehyde dibenzylacetal to give compound 11 and subsequent removal of the benzyl groupsby hydrogenation to give the aldehyde derivative compound 12. Theconjugation of compound 12 to KLH and HSA is performed through reductiveamination in the same way as for the preparation of conjugates 10a and10b, to provide the Man₉-KLH conjugate 13a and Man₉-HSA conjugate 13b,respectively.

EXAMPLE 2

As a crucial step to include the novel carbohydrate antigen into HIV-1vaccine design, the high-mannose oligosaccharide cluster is duplicatedthrough chemical synthesis. Assembly of the high-mannose oligosaccharidechains on a suitable scaffold molecule in a defined spatial orientationwould provide novel oligosaccharide clusters that mimic or capture theactual structure of the carbohydrate antigen as present on native HIV-1gp120. A general design of such a clustering antigenic structure isshown in FIG. 1, where four strands of the major HIV-1 high-mannose typeoligosaccharide, Man₉GlcNAc₂, are presented on a galactoside scaffold.Herein is disclosed an efficient synthesis of the tetravalenthigh-mannose oligosaccharide cluster and a related bivalentoligosaccharide cluster.

In recent years, many glycol-clusters were synthesized to study themultivalency and clustering effects in carbohydrate-protein interactions(71). But only a few involve the synthesis of glycol-clusters of largeoligosaccharide (73). To construct the designed high-mannoseoligosaccharide clusters, we took advantage of the highly efficientthiol-maleimide ligation reaction as the key step, which we haverecently exploited for the synthesis of very large and complexmultivalent peptides (74). The high-mannose type oligosaccharide foundon HIV-1, Man₉GlcNAc₂Asn 1, was prepared through digestion of soybeanagglutinin, which was isolated from soybean flour, according to thepublished method (75). The purified M₉GN₂Asn was identical to anauthentic sample and was further characterized by electrosprayionization mass

Spectroscopy (ESI-MS) [1998.73 (M+H)⁺999.69 9M+2H)²⁺, 918.65(M-Man+2H)²⁺, 837.68 (M-2Man+2H)²⁺, 756.70 (M-3Man+2H)²⁺, 675.52(M-4Man+2H)²⁺, 594.61 (M-5Man+2H)²⁺). For the ligation as shown in FIG.9, a sulfhydryl group was successfully introduced into theoligosaccharide in two steps (Scheme 1). First, the amino group inMan₉GlcNAc₂Asn (1) was selectively acylated with N-succinimidylS-acetylthioacetate (SATS)⁷⁵ in a phosphate buffer (pH 7.4) containing30% acetonitrile to give the N—(S-acetyl-thioacetyl) derivative (3)[ESI-MS: 2114.55 (M+H)⁺, 1057.66 (M+2H)²⁺, 976.55 (M-Man+2H)²⁺, 895.45(M-2Man+2H)²⁺, 815.54 (M-3Man+2H)²⁺, 733.43 (M-4Man+2H)²⁺, 652.39(M-5Man+2HI)²⁺, 571.34 (M-6Man+2H)²⁺]. As shown in FIG. 9, thethiol-protective group in compound 3 was then removed by treatment withhydroxylamine in a phosphate buffer (pH 7.5) to afford the thiolcompound 4, which was purified by HPLC and characterized by ESI-MS[2072.56 (M+H)⁺, 1036.71 (M+2H)²⁺, 955.68 (M-Man+2H)²⁺, 874.71(M-2Man+2H)²⁺, 793.66 (M-3Man+2H)²⁺, 712.56 (M-4Man+2H)²⁺, 631˜51(M-5Man+2H)²⁺, 550.66 (M-6Man+2H)²⁺]. The oligosaccharide derivative 4,which contains a sulfhydryl tag at the reducing terminus, is animportant intermediate for synthesizing useful glycol-clusters ofhigh-mannose type oligosaccharides.

A galactoside-based, tetravalent maleimide cluster 5 had been previouslysynthesized (73). The ligation between the maleimide cluster 5 and thethiol 4 was performed in a phosphate buffer (pH 6.6) containing 40%acetonitrile to afford the desired tetravalent oligosaccharide cluster 6(Scheme 2). Briefly, procedures for the preparation of theoligosaccharide cluster 6 are as follows. To a solution of thiol 4((7.60 mg, 3.67 umol) in a phosphate buffer (pH, 6.6, 50 mM, 1.2 ml) wasadded a solution of the galactose-based maleimide cluster 5 (shown inFIG. 10)(0.67 mg, 0.46 umol) in acetonitrile (0.8 ml). The mixture waskept at room temperature under nitrogen atmosphere. After 1 h. Themixture was lyophilized. The residue was purified by reverse-phase HPLCto afford the tetravalent high-mannose oligosaccharide cluster 6 (3.60mg, 81%). The purified product appeared as a single peak at 16.10 minunder the following analytical HPLC conditions: column, Waters Nova-PakC18 (3.9×150 mm); temperature, 40° C.; flow rate, 1 ml/min. The columnwas eluted with a linear gradient of acetonitrile (0-50%) containing0.1% TFA in 25 min.

HPLC revealed that the ligation was quantitative and was complete within1 h at room temperature. A simple HPLC purification gave the tetravalenthigh-mannose type oligosaccharide cluster 6 in 81% isolated yield. TheESI-MS and HPLC profile of compound 6 was shown in FIG. 12. Typicalfragments of compound 6 in ESI-MS are 2435.35 (M+4H)⁴⁺, 2394.95(M-Man+4H)⁴⁺, 1948.28 (M+5H)⁵⁺, 1915.85 (M-Man+5H)⁵⁺, and 1883.75(M-2Man+5H)⁵⁺, which are in agreement with its structure.

The synthetic approach should be equally efficient for constructing anarray of different oligosaccharide clusters on varied monosaccharides orother scaffolds. As another example, we synthesized a bivalenthigh-mannose oligosaccharide cluster 3 that will be useful forcomparative binding studies with the antibody 2G12. Thus, ligation ofthe thiol 4 and a bivalent scaffold 11-bis-maleimidetetraethyleneglycolBM(PBO)₄)(7) shown in FIG. 11, gave the bivalent oligosaccharide cluster8 in essentially quantitative yield. Compound 8 was purified by HPLC andcharacterized by ESI-MS [2248.78 (M+2H)²⁺, 1499A9 (M+3H)³⁺, 1445.45(M-Man+3H)³⁺, 1391.28 (M-2Man+3H)³⁺, 1337.44 (M-3Man+3H)³⁺, 1283.27(M-4Man+3H)³].

In summary, an efficient route for the construction of glycol-clustersinvolving large, native oligosaccharides is disclosed. The approachconsists of two key steps: selective introduction of a SH-tag into theoligosaccharide and a chemoselective ligation of the SH-taggedoligosaccharide with a maleimide cluster. The ligation reaction israpid, highly efficient, and essentially quantitative even when verylarge oligosaccharides are involved. A galactose-based, tetravalenthigh-mannose type oligosaccharide cluster (in which four strands of theoligosaccharide are arranged in a defined spatial orientation on thegalactose scaffold has been synthesized. The tetravalent oligosaccharidecluster provides a direct mimic to the carbohydrate epitope of thebroadly HIV-1 neutralizing antibody 2G12.

EXAMPLE 3

Methods and Materials

Materials:

Monosaccharides, pronase, Sephadex, trifluoroacetic acid, and reagentsfor ELISAs and buffers were purchased from Sigma-Aldrich and used asreceived. N-succinimidyl S-acetylthioacetate was from Pierce ChemicalCo. HPLC grade acetonitrile was purchased from Fisher Scientific. Theimmobilized endo-β-N-acetyl-glucosaminidase from Arthrobactor (Endo-A)was overproduced and purified according to the literature (79).

High-performance liquid chromatography (HPLC): Unless otherwisespecified, analytical HPLC was carried out on a Waters 626 HPLCinstrument under the following conditions: column, Waters Nova-Pak C18(3.9×150 mm); temperature, 40° C.; flow rate, 1 ml/min. The column waseluted with a linear gradient of acetonitrile (0-50%) containing 0.1%TFA in 25 min with UV detection at 214 nm. Preparative HPLC wasperformed on a Waters 600 HPLC instrument with a preparative C18 column(Waters Symmetry 300, 19×300 mm). The column was eluted with a suitablegradient of water-acetonitrile containing 0.1% TFA.

High-Performance Anion Exchange Chromatography Coupled with PulsedElectrochemical Detection (HPAEC-PED):

The analytical anion-exchange chromatography was performed on a DionexDX600 chromatography system (Dionex Corporation, Sunnyvale, Calif.)equipped with an electrochemical detector (ED50, Dionex Corporation,Sunnyvale, Calif.). The following conditions were used: column,CarboPac-PA1 (4×250 mm); Eluent A, 0.1 M NaOH; Eluent B, 1 M sodiumacetate (NaOAc) in 0.1 M NaOH; Gradient: 0-5 min, 0% B; 5-25 min, 0-15%B. Flow rate, 1 ml/min.

Competitive Enzyme-Linked Immunosorbent Assays (ELISAs):

Competitive ELISAs were performed to determine the relative inhibitionpotency of various carbohydrate antigens against the binding of 2G12 togp120. Microtiter plates were coated with human cell line 293-expressedHIV-1_(IIIB) gp120 (100 ng/ml) overnight at 4° C. After washing,non-specific binding was blocked with 5% BSA in PBS for 1 h at roomtemperature. The plates were then washed three times with 0.1%Tween-20/PBS. Serial dilutions (1:2) of various carbohydrate antigenswere mixed with an equal volume of MAb 2G12 (fixed final concentrationof 5 ng/ml) and added to the plates. The plates were incubated for 1 hat 37° C. and washed with washing buffer. To the plates was added a100-μl solution of 1:3000 diluted horseradish peroxidase-conjugated goatanti-human IgG in 0.5% BSA/PBS. After incubation for 1 h at 37° C., theplates were washed again and a 100-μl solution of 3,3′,5,5′-tetramethylbenzidine (TMB) was added. Color was allowed to develop for 5 min, andthe color reaction was quenched through adding a 100-μl solution of 0.5M H₂SO₄ to each well. The optical density was then measured at 450 nm.

Preparation of Homogeneous High-Mannose Type Oligosaccharides.

Man₉GlcNAc₂Asn and Man₉GlcNAc were prepared by enzymatic digestion ofsoybean agglutinin followed by chromatographic purification. Crudesoybean agglutinin (3.2 g) was obtained from 500 g of soybean flour(Sigma) through fractional precipitation with ammonium sulfate anddigested thoroughly with pronase (2×15 mg, Sigma) according to theliterature (74). The digestion was filtered and the filtrate waslyophilized. The residue was loaded onto a column (1.5×70 cm) ofSephadex G50 (Sigma), which was pre-equilibrated and eluted with 0.1MAcOH.

The fractions containing Man₉GlcNAc₂Asn were pooled and lyophilized. Thematerial was finally purified by reverse-phase HPLC to affordhomogeneous Man₉GlcNAc₂Asn (55 mg) as a white powder afterlyophilization. Treatment of Man₉GlcNAc₂Asn (20 mg) with immobilizedArthrobactor endo-β-N-acetylglucosaminidase (Endo-A) in an acetatebuffer (pH 6.0), followed by gel filtration on a column (1.5×50 cm) ofSephadex G25 gave pure Man₉GlcNAc (12 mg).

Homogeneous Man₅GlcNAc and Man₆GlcNAc were obtained from pronasedigestion of chicken ovalbumin followed by chromatographic purification.Chicken ovalbumin (Sigma) was digested with pronase to provide a crudemixture of Man₅- and Man₆-containing glycopeptides, according to theliterature (80). A crude glycopeptide (350 mg) was treated withimmobilized Endo-A to release Man₅GlcNAc and Man₆GlcNAc as a mixture.The two oligosaccharides were then separated by chromatography on acolumn (1×125 cm) of Celite-Charcoal (1:1, w/w), which was eluted by agradient of 0-20% aqueous ethanol to give pure Man₅GlcNAc (25 mg) andpure Man₆GlcNAc (30 mg). The purity of the above isolatedoligosaccharides was confirmed by HPAEC-PED and their identity wascharacterized by electron spray ionization mass spectrometry (ESI-MS).

Man₉GlcNAc₂Asn: HPAEC-PED, t_(R) 17.1 min; ESI-MS: calcd. forC₇₄H₁₂₄N₄O₅₈: 1997.77. Found: 1998.73 (M+H)⁺, 999.69 (M+2H)²⁺, 918.65(M-Man+2H)²⁺, 837.68 (M-2Man+2H)²⁺, 756.70 (M-3Man+2H)²⁺, 675.52(M-4Man+2H)²⁺, 594.61 (M-5Man+2H)²⁺.

Man₉GlcNAc: HPAEC-PED, t_(R) 16.9 min; ESI-MS, calcd. for C₆₂H₁₀₅NO₅₁:1679.57. Found: 1680.80 (M+H)⁺, 1518.64 (M-Man+H)⁺, 1356.72 (M-2Man+H)⁺,1194.54 (M-3Man+H)⁺, 1032.60 (M-4Man+H)⁺, 841.36 (M+2H)²⁺.

Man₆GlcNAc: HPAEC-PED, t_(R) 15.9 min; ESI-MS, calcd. for C₄₄H₇₅NO₃₆:1193.41. Found: 1216.84 (M+Na)⁺, 1194.81 (M+H)⁺, 608.99 (M+2Na)²⁺.

Man₅GlcNAc: HPAEC-PED, t_(R) 15.3 min; ESI-MS, calcd. for C₃₈H₆₅NO₃₁:1031.35. Found: 1054.70 (M+Na)⁺, 1032.79 (M+H)⁺, 528.07 (M+2Na)²⁺.

Preparation of the SH-Tagged Man₉ Oligosaccharide(Man₉GlcNAc₂Asn-Ac-SH).

To a solution of Man₉GlcNAc₂Asn (32 mg) in a phosphate buffer (3 ml, pH7.4) containing 20% acetonitrile was added a solution of N-succinimidylS-acetylthioacetate (20)(22 mg) in acetonitrile (0.5 ml). The mixturewas stirred at room temperature for 1 h and lyophilized. The product waspurified by reverse phase-HPLC to give the N—(S-acetyl-thioacetyl)Man9GlcNAc2Asn derivative (26 mg): analytical HPLC (gradient: 0-30%acetonitrile containing 0.1% TFA in 25 min; flow rate, 1 ml/min): t_(R)6.3 min; ESI-MS: 2114.55 (M+H)⁺, 1057.66 (M+2H)²⁺, 976.55 (M-Man+2H)²⁺,895.45 (M-2Man+2H)²⁺, 815.54 (M-3Man+2H)²⁺, 733.43 (M-4Man+2H)²⁺, 652.39(M-5Man+2H)²⁺, 571.34 (M-6Man+2H)²⁺].

A solution of the N—(S-acetyl-thioacetyl) derivative (20 mg) in aphosphate buffer (2 ml, 50 mM, pH 7.4) containing hydroxylamine (50 mM)was stirred at room temperature for 2 h, and the De-S-acetylated productwas directly purified by reverse phase HPLC to give the SH-taggedoligosaccharide Man₉GlcNAc₂Asn-Ac-SH (15 mg), which was characterized byHPLC and ESI-MS. Analytical HPLC (gradient: 0-30% acetonitrilecontaining 0.1% TFA in 25 min, flow rate, 1 ml/min): t_(R) 2.7 min;ESI-MS: 2072.56 (M+H)⁺, 1036.71 (M+2H)²⁺, 955.68 (M-Man+2H)²⁺, 874.71(M-2Man+2H)²⁺, 793.66 (M-3Man+2H)²⁺, 712.56 (M-4Man+2H)²⁺, 631.51(M5Man+2H)²⁺, 550.66 (M-6Man+2H)²⁺.

Synthesis of Tetravalent Oligomannose Cluster (Tetra-Man₉).

To a solution of Man₉GlcNAc₂Asn-Ac-SH (7.60 mg, 3.67 μmol) in aphosphate buffer (pH, 6.6, 50 mM, 1.2 ml) was added a solution of thegalactose-based maleimide cluster MC-1 (0.67 mg, 0.46 μmol) inacetonitrile (0.8 ml). The mixture was gently shaken at room temperatureunder nitrogen atmosphere for 1 h. The mixture was then lyophilized. Theligation product was purified by reverse-phase HPLC to afford Tetra-Man₉(3.60 mg, 81%). Analytical HPLC: t_(R), 16.1 min; ESI-MS: 2435.35(M+4H)⁴⁺, 2395.15 (M-Man+4H)⁴⁺, 1948.28 (M+5H)⁵⁺, 1915.85 (M-Man+5H)⁵⁺,and 1883.75 (M-2Man+5H)⁵⁺, which are in agreement with its structure.

Synthesis of Trivalent Oligomannose Cluster (Tri-Man₉).

The trivalent maleimide cluster MC-3 (1.0 mg) and Man₉GlcNAc₂Asn-Ac-SH(8.0 mg) were reacted in the same way as for the preparation ofTetra-Man₉. The ligation product was purified by reverse-phase HPLC togive the Tri-Man₉ (7.3 mg, 82%). Analytical HPLC, t_(R), 15.5 min;ESI-MS: 2457.90 (M+3H)³⁺, 1843.64 (M+4H)⁴⁺, 1802.92 (M-Man+4H)⁴⁺,1762.68 (M-2Man+4H)⁴⁺, 1722.10 (M-3Man+4H)⁴⁺, 1681.79 (M-4Man+4H)⁴⁺.

Synthesis of Bivalent Oligomannose Cluster (Bi-Man₉).

The bivalent maleimide cluster MC-2 (1.3 mg) and Man₉GlcNAc₂Asn-Ac-SH(9.4 mg) were reacted in the same way as for the preparation ofTetra-Man₉. The ligation product was purified by reverse-phase HPLC togive the Bi-Man₉ (6.1 mg, 80%). Analytical HPLC, t_(R), 15.4 min; ES-MS:2502.12 (M+2H)²⁺, 1668.22 (M+3H)³⁺, 1614.19 (M-Man+3H)³⁺, 1560.24(M-2Man+3H)³⁺, 1506.01 (M-3Man+3H)³⁺, 1452.54 (M-4Man+3H)³⁺.

Preparation of Man₉-dimer

Man₉GlcNAc₂Asn-Ac-SH (8 mg) was dissolved in a phosphate buffer (2 ml,50 mM, pH 7.5) and air was bubbled into the solution for 10 min. Thesolution was kept at room temperature overnight. The oxidized productthus formed was purified by reverse phase HPLC to give the Man₉-dimer(5.6 mg). Analytical HPLC (gradient: 0-30% acetonitrile containing 0.1%TFA in 25 min, flow rate, 1 ml/min): t_(R) 5.3 min; ESI-MS: 2072.0(M+2H)²⁺, 1381.6 (M+3H)³⁺, 1327.5 (M-Man+3H)³⁺, 1273.4 (M-2Man+3H)³⁺,1219.45 (M-3Man+3H)³⁺, 1165.41 (M-4Man+3H)³⁺, 1111.2 (M-5Man+3H)³⁺.

Binding of Homogeneous High-Mannose Type Oligosaccharides to 2G12

Structural analysis indicated that the high-mannose typeoligosaccharides on HIV-1 gp120 are heterogeneous, ranging from Man₅,Man₆, Man₇, Man₈, to Man₉ (81-83). However, isolation of individualhigh-mannose oligosaccharides directly from HIV-1 gp120 is technicallydifficult. To evaluate the affinity of each glycoform in 2G12interaction, we isolated three typical high-mannose typeoligosaccharides, namely Man₅GlcNAc, Man₆GlcNAc, and Man₉GlcNAc, asshown in FIG. 13 in high-purity from chicken ovalbumin and soybeanagglutinin, respectively. The mixture of Man₅GlcNAc and Man₆GlcNAcobtained by sequential treatment of chicken ovalbumin with pronase andArthrobactor endo-β-N-acetylglucosaminidase (Endo-A) was carefullyseparated on a Celite-Carbon chromatography to afford eacholigosaccharide. Based on HPAEC-PED analysis, the Man₅GlcNAc andMan₆GlcNAc thus isolated are at least 98% pure without crosscontamination (data not shown). Similarly, ultra-pure Man₉GlcNAc wasobtained through sequential digestion of soybean agglutinin with pronaseand Endo-A, followed by gel filtration on Sephadex G25 and reverse phaseHPLC purification.

The binding affinity of the high-mannose oligosaccharides was examinedby competitive inhibition of 2G12 binding to immobilized gp120, as shownin FIG. 14. The IC50 (concentration for 50% inhibition) for Man₉GlcNAc,Man₆GlcNAc, and Man₅GlcNAc were estimated to be 0.85, 70, and 200 mM,respectively. It should be pointed out that the solubility of Man₅GlCNAcand Man₆GlcNAc in aqueous solution is unexpectedly low (less than 80mM). As a result, the IC50 for Man₅GlcNAc and Man₆GlcNAc could not beaccurately determined. On a molar basis, the Man₉GlcNAc was 85-fold and244-fold more effective in inhibition of 2G12 binding than Man₆GlcNAcand Man₅GlcNAc, respectively. These results suggest that antibody 2G12preferably recognizes Man₉ moiety among the oligomannose glycoforms onHIV-1 gp120. The much higher affinity of Man₉GlcNAc to 2G12 than that ofMan₅GlcNAc and Man₆GlcNAc implicates the importance of terminalManα1,2Man linkages in antibody recognition. In comparison, Man₅GlcNAccontains three terminal Manα1,2Man linkages, Man₆GlcNAc contains oneManα1,2Man linkage, but Man₅GlcNAc does not have any terminal Manα1,2Manlinkage. The results are consistent with previous observation that theManα1,2Man moiety on HIV-1 gp120 is an essential component of 2G12epitope, as revealed by the binding of various glycosidase-treated gp120with 2G12 (83).

Design and Synthesis of Oligomannose Clusters as Mimics of 2G12 Epitope

The binding studies with homogeneous high-mannose type oligosaccharidesdemonstrated that the Man₉ subunit is preferred for 2G12 recognition. Asan important step to incorporate the novel epitope into HIV-1 vaccinedesign, the proposed oligomannose cluster was duplicated throughchemical synthesis. The assembly of oligomannose such as Man₉ on asuitable scaffold molecule was generated to provide oligosaccharideclusters that may mimic or capture 2G12 epitope as present on HIV-1gp120. Bi-, tri- and tetra-valent Man₉ clusters were synthesized basedon a galactopyranoside scaffold as shown in FIG. 15. Compared to othertypes of molecules, monosaccharides have several advantages to serve asa scaffold. They have a rigid ring structure, possess multiplefunctionalities, and provide a defined three-dimensional spatialarrangement of substituents. When a galactopyranoside is used as thescaffold to present the oligosaccharides, the oligosaccharide chainsbeing installed at C-3, 4, and 6 positions will face up above the sugarring to form a cluster, while the oligomannose sugar chain at positionC-2 is likely to be located on the flank of the cluster. Thisarrangement was determined to mimic the spatial orientation of thecarbohydrate epitope of antibody 2G12. Based on the reported structure(21) of gp120 core with remodeled N-glycans, the distances between theasparagines (Asn) side chains of the pairs N295-N332, N332-N392, andN295-N392 are estimated to be 5.8, 20.3, and 23.6 Å, respectively. AMan₉GlcNAc₂Asn moiety was positioned on a synthesized galactose-basedmaleimide cluster previously synthesized by the current inventor (83).It was found that the maleimide cluster can host four Man₉GlcNAc₂Asnmoieties, in which the distances among the Asn residues are in the rangeof 8-30 Å (data not shown).

The key step in the synthesis is the chemoselective, maleimidecluster-thiol ligation reaction, which was recently exploited for thesynthesis of large multivalent peptides and glycoconjugates (83-84). Tointroduce a sulfhydryl (SH)-tag into the oligomannose moiety, the freeamino group in Man₉GlcNAc₂Asn was first acylated with N-succinimidylS-acetylthioacetate (SATS)(86) to give the N—(S-acetyl-thioacetyl)derivative. The S-acetyl group was then removed selectively by treatmentwith hydroxylamine to afford the SH-containing oligosaccharide,Man₉GlcNAc₂Asn-Ac-SH. The synthesis of the tetravalent maleimide clusterMC-1 was previously reported. The bi- and tri-valent maleimide clusterMC-2 and MC-3 were synthesized in a similar way starting with modifiedgalactoside scaffold (Details of the synthesis will be reportedelsewhere). Chemoselective ligation between the Man₉GlcNAc₂Asn-Ac-SH andthe maleimide cluster MC-1 was performed in a phosphate buffer (pH 6.6).HPLC monitoring indicated that the ligation was quantitative and wascomplete within 1 h at room temperature. Simple reverse phase HPLCpurification gave the tetravalent oligomannose cluster Tetra-Man₉ in 81%yield. The structure of Tetra-Man₉ was characterized by electron sprayionization-mass spectroscopy (ESI-MS) (FIG. 12). The ESI-MS spectrumrevealed typical signals at 2435.35 (M+4H)⁴⁺, 2395.15 (M-Man+4H)⁴⁺,1948.28 (M+5H)⁵⁺, 1915.85 (M-Man+5H)⁵⁺, and 1883.7 (M-2Man+5H)⁵⁺, whichare in agreement with the structure.

Similarly, the bi- and trivalent Man₉ clusters, Bi-Man₉ and Tri-Man₉,were synthesized through ligation of Man₉GlcNAc₂Asn-Ac-SH with themaleimide clusters MC-2 and MC-3, respectively. On the other hand, adimmer of Man₉GlcNAc₂Asn was prepared through oxidation ofMan₉GlcNAc₂Asn-Ac-SH to give the Man₉-dimer (FIG. 15). All the finalproducts were purified by HPLC to homogeneity and characterized byESI-MS.

Binding of the Synthetic Man₉-clusters to 2G12

The synthetic Man₉-clusters were examined for competitive inhibition of2G12 binding to immobilized gp120 (FIG. 16). A significant clusteringeffect was observed for the Man₉-clusters as shown in Table 1 below.

Potency on carbohydrate inhibition of 2G12 binding to gp120

IC 50 Relative Affinity Carbohydrate antigens (nM) Molar basisValency-corrected Man₅GlcNAc 200 estimated 0.004 0.004 Man₆GlcNAc 700.012 0.012 Man₉GlcNAc 0.98 0.84 0.84 Man₉GlcNAc2Asn 0.82 1.0 1.0Man9-dimer 0.40 2.1 1.0 Bi-Man9 0.13 6.3 3.2 Tri-Man9 0.044 18.6 6.2Tetra-Man9 0.013 63.1 15.8

If IC₅₀ is taken as an indication for relative affinity (Table 1), theTetra-Man₉ was found to inhibit the 2G12 binding 63-fold moreeffectively than monomeric Man₉GlcNAc₂Asn does on a molar basis. Thiscorresponds to a 16-fold increase in the affinity to 2G12 for eacholigosaccharide subunit in Tetra-Man₉ on a valence-corrected basis, whencompared with monomeric Man₉. On the other hand, the trivalent clusterTri-Man₉ was 19-fold (on a molar basis) or 6-fold (on avalence-corrected basis) more effective than Man₉GlcNAc₂Asn ininhibition of 2G12 binding to gp120. Interestingly, for the two bivalentoligosaccharides Bi-Man₉ and Man₉-dimer, they showed significantlydifferent affinity toward 2G12. The Man₉-dimer inhibited the2G12-binding 2-fold more effectively than Man₉GlcNAc₂Asn, while theBi-Man₉ was 6-fold better than Man₉. This suggests that the geometry andthe distance between the two oligomannose subunits are important factorsin controlling antibody recognition. It was also found that the subunitMan₉GlcNAc and Man₉GlcNAc₂Asn showed essentially the same affinity for2G12 binding. The data suggest that the GlcNAc-Asn moiety linking theoligosaccharide to the protein is not directly involved in therecognition with 2G12. The observation could not be revealed throughmutagenesis studies.

The 2G12 binding studies demonstrated that Man₉GlNAc is 85- and 244-foldmore effective than Man₆GlcNAc and Man₅GlcNAc, respectively, ininhibition of 2G12 binding to gp120. Therefore, oligomannose Man₉ shouldbe the “building block” of choice for creating mimics of 2G12's epitope.The established scaffold approach of the present invention allowsefficient synthesis of template-assembled oligosaccharide clusters, inwhich the oligomannose sugar chains are presented in a definedthree-dimensional fashion. Thus, bi-, tri-, and tetra-valentoligomannose clusters were efficiently constructed on a galactosescaffold, using the chemoselective maleimide cluster-thiol ligation asthe key step.

An apparent clustering effect of the oligomannose clusters was observedin the inhibition studies. The tetra-, tri-, and bi-valent oligomannoseclusters are 63-, 19-, and 6-fold more effectively than the monomericMan₉GlcNAc₂Asn in inhibition of 2G12 binding to gp120 on a molar basis.The enhanced affinity for the clusters with higher valency suggests thatantibody 2G12 may have multiple binding sites for the carbohydrateantigen. The observed enhancement in 2G12 binding for the higher-valentoligomannose clusters is consistent with the existence of additionalbinding sites on 2G12 for carbohydrate antigen. Another interestingfinding in the above reported binding studies came from the two bivalentoligomannose compounds, Bi-Man₉ and Man₉-dimer. They showedsignificantly different binding potency to 2G12 despite the samevalency. The Bi-Man₉ is 3-fold more effective than Man₉-dimer ininhibition of 2G12 binding to gp120. The results suggest that thecontrol of geometry and distance of the subunits is important to achievea tight multivalent interaction between the carbohydrate antigen and theantibody. As such modification and manipulation of the spatialorientation of oligomannose sugar chains on the scaffold provides forimproved epitope mimics and increase affinity of gp120 to the epitopemimics relative to 2G12.

REFERENCES

All publications mentioned herein are hereby incorporated by referenceherein for the all purposes.

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1. A high-mannose oligosacoharide cluster that mimics a carbohydrateepitope on gp120 comprising four high-mannose oligosaccharidespositioned on a cyclic core scaffolding framework, wherein the cycliccore scaffolding framework comprises monosaccharides, or cyclic organiccompounds, wherein the four high-mannose oligosaccharides are Man₉, andwherein the positioning of high-mannose oligosaccharides on the cycliccore scaffolding framework mimics the carbohydrate epitope on gp120having affinity for 2G12 antibodies.
 2. The high-mannose oligosaccharidecluster according to claim 1, further comprising an immunogenic proteinconjugated to the high-mannose oligosaccharide cluster thereby producinga high-mannose oligosaccharide/protein cluster.
 3. The high-mannoseoligosacoharide cluster of claim 2, wherein the immunogenic protein isselected from the group consisting of keyhole limpet hemocyanin, tetanustoxoid, diphtheria toxoid, bovine serum albumin, ovalbumin,thyroglobulin, myoglobin, cholera toxin β-subunit, immunoglobulin and/ortuberculosis purified protein derivative.
 4. The high-mannoseoligosaccharide cluster of claim 2 comprising four Man₉ covalentlyattached to a galactose scaffolding framework, wherein the immunogenicprotein comprises keyhole limpet hemocyanin.
 5. A pharmaceuticalcomposition comprising the high-mannose oligosaccharide cluster ofclaim
 1. 6. A method for generating a high-mannose oligosaccharidecluster, the method comprising: covalently attaching four high-mannoseoligosaccharide chain to a cyclic core scaffolding framework, whereinthe cyclic core scaffolding framework comprises monosaccharides, orcyclic organic compounds, wherein the four high-mannose oligosaccharidesare Man₉, and wherein the positioning of high-mannose oligosaccharideson the cyclic core scaffolding framework mimics a carbohydrate epitopeon gp120 having affinity for 2G12 antibodies.
 7. The method according toclaim 6, wherein the high-mannose oligosaccharide chain is extractedfrom the digestion of soybean agglutinin or produced by chemicalsynthesis.
 8. The method of claim 6, further comprising conjugating animmunogenic protein to the high-mannose oligosaccharide cluster.
 9. Amethod of inducing production of HIV antibodies that exhibit affinityfor a conserved cluster of oligomannose sugars on gp120, the methodcomprising: administering to an animal the high-mannose oligosaccharideaccording to claim 2 in an amount sufficient to induce production ofantisera specific for the high-mannose oligosaccharide; and collectingthe antisera.
 10. A method for detecting candidate compounds thatpotentially interact with a conserved cluster of oligomannose sugars ongp120, the process comprising: contacting the candidate compound withthe high-mannose oligosaccharide cluster according to claim 1; anddetermining the binding affinity of the candidate compound forhigh-mannose oligosaccharide cluster.